真菌感染檢查課件

1、第四節(jié) 真菌感染檢查第四節(jié) 真菌感染檢查檢驗特點形態(tài)學檢查為檢測真菌的重要手段抗原檢測適合血清和腦脊液中隱球菌、念珠菌、莢膜組織胞漿菌。血清學診斷適用于深部真菌感染。檢驗特點形態(tài)學檢查為檢測真菌的重要手段檢測程序標本直接鏡檢抗原檢出分離培養(yǎng)不染色乳酸酚棉藍染色墨汁染色氫氧化鉀消化后涂片鏡檢二相性真菌腦心浸液腦心浸液沙氏培養(yǎng)基病原性真菌血瓊脂平板觀察菌落性狀和菌絲孢子形態(tài)檢測程序標本直接鏡檢抗原檢出分離培養(yǎng)不染色乳酸酚棉藍染色墨汁真菌感染檢查課件真菌的菌落-真菌分類重要的依據(jù) 真菌的菌落一般是指在一定基質(zhì)上，接種某種真菌的一個孢子或一段菌絲，經(jīng)過培養(yǎng)，向四周蔓延生長出絲狀的群體稱為真菌菌落。菌落

2、呈放射狀生長，因而菌落外周的生命力最旺盛。在不同成分的培養(yǎng)基上和不同條件下培養(yǎng)，同一種真菌，形成的菌落也有差別。因此，菌落的形態(tài)觀察是指在固定的條件下，菌落所表現(xiàn)的形狀、大小、色澤和結構等。不同的真菌菌落所表現(xiàn)特征差別顯著，它為真菌分類工作提供了重要的依據(jù)。真菌的菌落-真菌分類重要的依據(jù) 真菌的菌落一般菌落顏色除酵母菌的菌落比較簡單，絲狀真菌的菌落形態(tài)各種各樣。常以下述特征加以描述。其顏色的多樣很難用色澤描述，常借助于色譜加以鑒別。很多真菌產(chǎn)生多種顏色的色素，使菌落的背面染有顏色，有的甚至分泌可溶性色素，擴散到全部基質(zhì)中去。菌落顏色除酵母菌的菌落比較簡單，絲狀真菌的菌落形態(tài)各種各樣。菌落質(zhì)地氣

3、生菌絲構成表面菌絲體質(zhì)地。底部菌絲體埋伏于培養(yǎng)基中或緊貼培養(yǎng)基表面向四周蔓延，由底部體直接生出分生孢子梗，菌落外觀呈短茸毛狀，由底部菌絲體生出氣生菌絲，再由它生出分生孢子梗，因而往往有纏繞的氣生菌絲團，一般菌落較厚，呈絮狀。也有部分氣生菌絲扭結成繩索、還有分生孢子梗自底部菌絲成束地生長，在菌落的外觀上成粒狀或粉狀。有些真菌產(chǎn)生子實體或菌核，則菌落表面呈顆粒結構。有的菌落出現(xiàn)同心環(huán)或輻射狀溝紋。有的真菌只在菌落中間區(qū)域生出分生孢子頭，邊緣菌絲則不生育。菌落的大小也大有不同，有些種的菌落可蔓延擴展到整個培養(yǎng)基，而另一些種的菌落則局限生長。菌落質(zhì)地氣生菌絲構成表面菌絲體質(zhì)地。底部菌絲體埋伏于培養(yǎng)基中

4、真菌菌落外觀結構描述菌落表面平滑或具皺紋、致密或疏松、同心環(huán)或輻 射狀溝紋等；菌落質(zhì)地絨毛狀、氈狀、棉絮狀、羊毛狀、束狀、繩索狀、粉粒狀、明膠狀或皮革狀等；菌落的邊緣可呈全緣、鋸齒狀、樹枝狀或纖毛狀等；菌落高度扁平、丘狀隆起、中心部分突起或凹陷等。 真菌菌落外觀結構描述菌落表面平滑或具皺紋、致密或疏松、同真菌感染檢查課件193 patients (392 samples); 43 with ICSensitivity SpecificityMannan (ag)40 %98 %Antimannan (ab)53 %94 %ag and/or ab80 %93 % （一）抗原：甘露聚糖Detec

5、tion of Mannan/Antimannan in Serum of Patients with Invasive CandidiasisSendid B et al, J Clin Microbiol 1999;37:1510-7 （一）抗原：甘露聚糖Detection of Manna（二）抗原：半乳甘露聚糖Serial Aspergillus Galactomannan Screeningn = 362 high-risk neutropenic patientsSandwich ELISA 2 times weekly, 11.7% positiven = 30 proven I

6、An = 9 probable IAn = 264 with no IASensitivity89.7%Specificity98.1%PPV87.5%NPV98.4%Maertens J et al, Blood 2001;97:1604-10（二）抗原：半乳甘露聚糖Serial AspergilluGM抗原檢測用于侵襲性曲霉病診斷與臨床診斷和療效反應有良好的一致性。檢測方法有ELISA、放射免疫分析（RIA）和乳膠凝集試驗等，但敏感性低。ELISA檢測24只兔(王莉，等。2003 ) 敏感性和特異性： 血清 95% 78% 尿液 90% 51.4%GM抗原檢測用于侵襲性曲霉病診斷與臨床診斷

7、和療效反應有良好的GM抗原ELISA檢測方法（Platelia試劑盒）48例患者：確診IA 4例，3例陽性；高度懷疑IA 13例，8例陽性；可疑IA 31例，2例陽性。 王莉，等。臨床皮膚科雜志，待發(fā)表。靈敏性為64.7%特異性為93.5%假陽性率為6.5%假陰性率為35.3%GM抗原ELISA檢測方法（Platelia試劑盒）48例（四）-1,3葡聚糖使用儀器及配套試劑盒能快速地檢測出血液和體液中真菌-1,3葡聚糖的實際含量，對真菌的早期診斷具有重要意義。 （四）-1,3葡聚糖使用儀器及配套試劑盒能快速地檢測出血液-1,3-D-葡聚糖檢測陰性是否需要作進一步的復查？ Furuya的報道即使是

8、確診的深部真菌感染患者,-1,3-D-葡聚糖檢測也有可能是陰性,因此單次-1,3-D-葡聚糖檢測陰性并不能排除深部真菌感染的可能，必須在疾病的過程中重復進行-1,3-D-葡聚糖檢測。 Furuya T et al. Usefulness of -D-glucan measurement for diagnosis of deep mycosis, Jnp J Antibiot 1993; 46(6):437-443-1,3-D-葡聚糖檢測陰性是否需要作進一步的復查？ 真菌分子生物學的鑒定方法核酸堿基GC比分析限制性片段長度多態(tài)性Southern印跡分析脈沖場凝膠電泳（PFGE）PCR隨機擴增多

9、態(tài)性 (RAPD)DNA片段測序等真菌分子生物學的鑒定方法核酸堿基GC比分析真菌感染檢查項目的選擇和應用淺部真菌直接顯微鏡檢查深部真菌標本培養(yǎng)、觀察培養(yǎng)物性狀并借助理化特性作鑒別。真菌感染檢查項目的選擇和應用淺部真菌直接顯微鏡檢查Direct Examination It is highly recommended that a direct microscopic examination be made on most this provide an immediate presumptive diagnosis for the physician, but it may also aid

10、in the selection of an appropriate culture medium. A phase-contrast microscope is a valuable adjunct in the direct examination of specimens. The advantages include the following:1)mounts can be made and examined quickly;2)there is no need for direct staining;and3)the objects can be clearly visualize

11、d.Direct Examination It is Tests for detection of fungal antibodies(1)Determination of antibody and/or antigen titers may be useful in diagnosing fungal infections and when performed in a serial fashion also provide a means of monitoring the progression of disease and the patients response to therap

12、y .Tests for detection of fungal With the exceptionof antibody tests for histoplasmosis and occidioidomycosis, however,most tests designed to detect an antibody response as a means of diagnosing invasive fungal infections lack sensitivity and specificity,are poorly standardized and are not widely av

13、ailableTests for detection of fungal antibodies(2)With the exceptionof antibodyTests for detection of fungal antibodies(3)Antibody tests for Candida and Aspergillus. Are often unable to distinguish active from past infection and Colonization from transient fungemiaTests for detection of fungal Tests

14、 for detection of fungal antibodies(4) In addition,a negative test for fungus-specific antibodies does not rule out infection because imunocompromised patients, and some individuals with disseminated infection may not mount an antibody response to the infecting organism .Tests for detection of funga

15、l Tests for detection of fungal antigens(1) Detection of fungal antigens or metabolites in serum or other body fluids represents the most direct means of providing a serologic diagnosis of invasive fungal infection .Both the RIA and the EIA tests have been shown to be rapid,sensitive, specific, rapi

16、d ,and reproducible. Tests for detection of fungal Tests for detection of fungal antigens(2)Although significant advances have been made in recent years,most methods for rapid detection of fungal antigens are available only in research laboratories .Notable exceptions are the tests for detection of

17、the polysaccharid ride antigens of C.neoformans and H.capsulatumd .Tests for detection of fungal Tests for detection of fungal -specific nucleic acid sequences(1)The use of the PCR to detect fungal nucleic acids directly in clinical material offers great promise for the rapid diagnosis of fungal infections .Tests for detection of funga